Dabigatran (DABI) is an oral anticoagulant and a reversible direct inhibitor of thrombin (DTI). Thrombin is known to induce the activation of platelets to aggregate by binding to and cleaving the extracellular <i>N</i>-terminal domains of protease-activated receptors 1 and 4 (PAR1 and PAR4). Recently, it has been shown that DABI can inhibit platelet aggregation through a direct effect on platelet thrombin receptors (PAR-4), which is correlated with a reduced expression of GPIIb/IIIa, CD63, and P-selectin on platelets after dabigatran treatment. The research presented herein further explored the molecular and cellular pathways modulated by DABI in the thrombin activated human platelets from healthy donors by employing label-free proteomics approaches. We hypothesized that a global proteomics analysis of thrombin activated human platelets treated with DABI would reveal the integrin-linked kinase (ILK) and integrin mediated signaling as major significantly down-regulated pathways by DABI. To test our hypothesis, we monitored changes in the protein expression profiles of platelets, purified from healthy human donors and <i>ex-vivo </i>activated with thrombin in presence or absence of DABI (500 ng/ml). The label free proteomics platform employed the nanoLC-ESI MS/MS sequencing of tryptic/Glu-C/Lys-C generated peptides from total platelets lysates, as well as a Q-Exactive quadrupole orbitrap mass spectrometer coupled with the label free quantification (LFQ) module provided by PEAKS 8.5 software. The quantitative analysis of the biochemical pathways was accomplished with ingenuity pathway analysis using the protein ratios extracted from LFQ analyses. The bioinformatics analysis predicted that many proteins involved in the integrin signaling pathways, the actin-mediated cell signaling and platelets aggregation, were at least two-fold down-regulated (p&lt;0.05) in the DABI-treated platelets than in the control, untreated, and thrombin activated platelets. Our results further advocate for the implementation of platelets proteomics profiling as a reliable assay for monitoring the efficacy of selected drug treatment during Acute Coronary Syndrome (ACS) management.

Pharmacoproteomic analysis highlights the dabigatran induced downregulation of integrin triggered signaling and inhibition of aggregation in thrombin activated human platelets

256th National Meeting (2018)

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